Recently, a large number of measurements using intermolecular interactions such as immune responses are being carried out in clinical tests, etc. However, since conventional methods require complicated operations or labeling substances, several techniques are used that are capable of detecting the change in the binding amount of a test substance with high sensitivity without using such labeling substances. Examples of such a technique may include a surface plasmon resonance (SPR) measurement technique, a quartz crystal microbalance (QCM) measurement technique, and a measurement technique of using functional surfaces ranging from gold colloid particles to ultra-fine particles. The SPR measurement technique is a method of measuring changes in the refractive index near an organic functional film attached to the metal film of a chip by measuring a peak shift in the wavelength of reflected light, or changes in amounts of reflected light in a certain wavelength, so as to detect adsorption and desorption occurring near the surface. The QCM measurement technique is a technique of detecting adsorbed or desorbed mass at the ng level, using a change in frequency of a crystal due to adsorption or desorption of a substance on gold electrodes of a quartz crystal (device). In addition, the ultra-fine particle surface (nm level) of gold is functionalized, and physiologically active substances are immobilized thereon. Thus, a reaction to recognize specificity among physiologically active substances is carried out, thereby detecting a substance associated with a living organism from sedimentation of gold fine particles or sequences.
In all of the above-described techniques, the surface where a physiologically active substance is immobilized is important. Surface plasmon resonance (SPR), which is most commonly used in this technical field, will be described below as an example.
A commonly used measurement chip comprises a transparent substrate (e.g., glass), an evaporated metal film, and a thin film having thereon a functional group capable of immobilizing a physiologically active substance. The measurement chip immobilizes the physiologically active substance on the metal surface via the functional group. A specific binding reaction between the physiological active substance and a test substance is measured, so as to analyze an interaction between biomolecules.
A method for producing a hydrogel has been disclosed in detail in, for example, JP Patent No. 2815120, as a detection surface having a functional group capable of immobilizing a physiologically active substance thereon. Specifically, a layer of 16-mercaptohexadecanol is bound to a gold film to thereby form a barrier layer. On this gold film, hydroxyl groups on the barrier layer are treated with epichlorohydrin and thereby epoxy-activated. In a subsequent step, dextran is attached to the barrier layer via ether linkages. The dextran matrix is next reacted with bromoacetic acid to thereby introduce carboxymethyl groups therein.
The following approach has been disclosed as an approach for immobilizing a physiologically active substance (e.g., a protein or amino acid) having an amino group into the carboxymethyl-modified dextran surface produced based on this procedure: a part of the carboxyl groups in the carboxymethyl-modified dextran are modified so as to give reactive ester function, for example, by treatment with an aqueous solution of N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) hydrochloride. The residual charges, that is, unreacted carboxyl groups, will contribute to effecting the concentration of the physiologically active substance on the detection surface. Such a detection surface can be brought into contact with an aqueous solution of the physiologically active substance (protein or amino acid) containing an amino group to thereby covalently bond the physiologically active substance containing an amino group to the dextran matrix.
The hydrogel produced by the aforementioned method is capable of three-dimensionally immobilizing the physiologically active substance containing an amino group, and therefore exhibits excellent performance as a detection surface of a biosensor. However, the method for producing a hydrogel using the procedures described above is complicated and requires long production time and the use of compounds such as epichlorohydrin and bromoacetic acid. Therefore, it presented a safety problem.